Methods of preparing cytotoxic benzodiazepine derivatives

ABSTRACT

The invention provides novel methods for preparing indolinobenzodiazepine dimer compounds and their synthetic precursors.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/878,991, filed Jan. 24, 2018, which claims the benefit of the filingdate, under 35 U.S.C. § 119(e), of U.S. Provisional Application No.62/450,270, filed on Jan. 25, 2017, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel methods for preparing cytotoxicindolinobenzodiazepine derivatives.

BACKGROUND OF THE INVENTION

It has been shown that cell-binding agent conjugates ofindolinobenzodiazepine dimers that have one imine functionality and oneamine functionality display a much higher therapeutic index (ratio ofmaximum tolerated dose to minimum effective dose) in vivo compared topreviously disclosed benzodiazepine derivatives having two iminefunctionalities. See, for example, WO 2012/128868. The previouslydisclosed method for making the indolinobenzodiazepine dimers with oneimine functionality and one amine functionality involves partialreduction of indolinobenzodiazepine dimers having two iminefunctionalities. The partial reduction step generally leads to theformation of fully reduced by-product and unreacted starting material,which requires cumbersome purification step and results in low yield.

Thus, there exists a need for improved methods for preparing theindolinobenzodiazepine dimers that are more efficient and suitable forlarge scale manufacturing process.

SUMMARY OF THE INVENTION

The present invention provides modular synthetic methods for preparingindolinobenzodiazepine dimer compounds and their synthetic precursors.Compared to the previously disclosed methods, the methods of the presentinvention are modular and more versatile as well as suitable for largescale manufacturing process.

In one embodiment, the present invention provides a method of preparinga compound of formula (A):

or a salt thereof, comprising reacting a compound of formula (V):

or a salt thereof, with a compound of formula (X):

wherein:

each double line

between N and C independently represents a single bond or a double bond,provided that when it is a double bond X is absent and Y is —H, and whenit is a single bond, X and Y are both —H; and

E is —OH, halide or —C(═O)E is an activated ester.

In another embodiment, the present invention provides a method ofpreparing a compound of formula (A):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (IV):

or a salt thereof, with a reducing agent to form a compound of formula(V):

or a salt thereof; and

2) reacting the compound of formula (V) or a salt thereof, with acompound of formula (X):

wherein:

each double line

between N and C independently represents a single bond or a double bond,provided that when it is a double bond X is absent and Y is —H, and whenit is a single bond, X and Y are both —H; and

E is —OH, halide or —C(═O)E is an activated ester.

Also provided in the present invention is a method of preparing acompound of formula (Xa):

or a salt thereof, comprising reacting the compound of formula (IX):

or a salt thereof, with a carboxylic acid deprotecting agent, wherein P₁is a carboxylic acid protecting group.

In another embodiment, the present invention is directed to a method ofpreparing a compound of formula (IX):

comprising reacting a compound of formula (VIII):

or a salt thereof, with a compound of formula (c):

or a salt thereof, wherein E₁ is —OH, halide or —C(═O)E₁ is an activatedester; and P₁ is a carboxylic acid protecting group.

In yet another embodiment, the present invention provides a method ofpreparing a compound of formula (Xa):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (VIII):

or a salt thereof, with a compound of formula (c):

or a salt thereof, to form a compound of formula (IX):

and

2) reacting the compound of formula (IX) with a carboxylic aciddeprotecting agent, wherein E₁ is —OH, halide or —C(═O)E₁ is anactivated ester; and P₁ is a carboxylic acid protecting group.

Also provided in the present invention is a method of preparing acompound of formula (II),

comprising reacting a compound of formula (I):

with hydrochloric acid in toluene.

In another embodiment, the present invention is directed to a method ofpreparing a compound of formula (IV):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (I):

with hydrochloric acid in toluene to form a compound of formula (II):

2) reacting the compound of formula (II) with a monomer compound offormula (a),

to form a compound of formula (III):

or a salt thereof;

3) reacting the compound of formula (III) or a salt thereof with amonomer compound of formula (b):

to form the compound of formula (IV) or a salt thereof.

In another embodiment, the present invention provides a method ofpreparing a compound of formula (A-1):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (I):

with hydrochloric acid in toluene to form a compound of formula (II):

2) reacting the compound of formula (II) with a monomer compound offormula (a),

to form a compound of formula (III):

or a salt thereof;

3) reacting the compound of formula (III) or a salt thereof with amonomer compound of formula (b):

to form a compound of formula (IV-1):

or a salt thereof;

4) reacting the compound of formula (IV) or a salt thereof with areducing agent to form a compound of formula (V-1):

or a salt thereof; and

5) reacting the compound of formula (V-1) or a salt thereof, with acompound of formula (X-1):

to form the compound of formula (A-1) or a salt thereof, wherein E is—OH, halide or —C(═O)E is an activated ester.

The present invention also provide compounds described herein, such ascompounds of formula (VII), (VIII), (VIIIa), (IX), (IX-1), (X), (Xa),(X-1) or (X-1a) or a salt thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1-3 show proton NMR spectra of the compounds of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention.

It should be understood that any of the embodiments described herein canbe combined with one or more other embodiments of the invention, unlessexplicitly disclaimed or improper. Combination of embodiments are notlimited to those specific combinations claimed via the multipledependent claims.

Definitions

“Alkyl’ as used herein refers to a saturated linear or branchedmonovalent hydrocarbon radical. In preferred embodiments, a straightchain or branched chain alkyl has thirty or fewer carbon atoms (e.g.,C₁-C₃₀ for straight chain alkyl group and C₃-C₃₀ for branched alkyl),and more preferably twenty or fewer carbon atoms. Even more preferably,the straight chain or branched chain alkyl has ten or fewer carbon atoms(i.e., C₁-C₁₀ for straight chain alkyl group and C₃-C₁₀ for branchedalkyl). In other embodiments, the straight chain or branched chain alkylhas six or fewer carbon atoms (i.e., C₁-C₆ for straight chain alkylgroup or C₃-C₆ for branched chain alkyl). Examples of alkyl include, butare not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl,2-methyl-1-propyl, —CH₂CH(CH₃)₂), 2-butyl, 2-methyl-2-propyl, 1-pentyl,2-pentyl 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl,2-methyl-1-butyl, 1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2-pentyl,3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl,2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl,1-octyl, and the like. Moreover, the term “alkyl” as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. As used herein,(C_(x)-C_(xx))alkyl or C_(x-xx)alkyl means a linear or branched alkylhaving x-xx carbon atoms.

The term “aryl” as used herein, include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. Aryl groups include, but are not limited to, phenyl,phenol, aniline, and the like. The terms “aryl” also includes“polycyclyl”, “polycycle”, and “polycyclic” ring systems having two ormore rings in which two or more atoms are common to two adjoining rings,e.g., the rings are “fused rings,” wherein at least one of the rings isaromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, or aromatic rings. In some preferredembodiments, polycycles have 2-3 rings. In certain preferredembodiments, polycyclic ring systems have two cyclic rings in which bothof the rings are aromatic. Each of the rings of the polycycle can besubstituted or unsubstituted. In certain embodiments, each ring of thepolycycle contains from 3 to 10 carbon atoms in the ring, preferablyfrom 5 to 7. For example, aryl groups include, but are not limited to,phenyl (benzene), tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, andnaphthyl, as well as benzo-fused carbocyclic moieties such as5,6,7,8-tetrahydronaphthyl, and the like. In some embodiments, the arylis a single-ring aromatic group. In some embodiments, the aryl is atwo-ring aromatic group. In some embodiments, the aryl is a three-ringaromatic group.

The term “heteroaryl” as used herein, refers to substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom (e.g., O, N, or S),preferably one to four or one to three heteroatoms, more preferably oneor two heteroatoms. When two or more heteroatoms are present in aheteroaryl ring, they may be the same or different. The term“heteroaryl” also includes “polycyclyl”, “polycycle”, and “polycyclic”ring systems having two or more cyclic rings in which two or more ringatoms are common to two adjoining rings, e.g., the rings are “fusedrings,” wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaromatics, and/or heterocyclyls. In some preferredembodiments, polycyclic heteroaryls have 2-3 rings. In certainembodiments, preferred polycyclic heteroaryls have two cyclic rings inwhich both of the rings are aromatic. In certain embodiments, each ringof the polycycle contains from 3 to 10 atoms in the ring, preferablyfrom 5 to 7 atoms in the ring. For examples, heteroaryl groups include,but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrazine, pyridazine, quinoline,pyrimidine, indolizine, indole, indazole, benzimidazole, benzothiazole,benzofuran, benzothiophene, cinnoline, phthalazine, quinazoline,carbazole, phenoxazine, quinoline, purine and the like. In someembodiments, the heteroaryl is a single-ring aromatic group. In someembodiments, the heteroaryl is a two-ring aromatic group. In someembodiments, the heteroaryl is a three-ring aromatic group.

The heteroaryl groups can be carbon (carbon-linked) or nitrogen(nitrogen-linked) attached where such is possible. By way of example andnot limitation, carbon bonded heteroaryls are bonded at position 2, 3,4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine,position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of apyrazine, position 2, 3, 4, or 5 of a furan, thiofuran, thiophene, orpyrrole, position 2, 4, or 5 of an oxazole, imidazole or thiazole,position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8of an isoquinoline.

By way of example and not limitation, nitrogen bonded heteroaryls arebonded at position 1 of a pyrrole, imidazole, pyrazole, indole,1H-indazole, position 2 of a isoindole, and position 9 of a carbazole,or β-carboline.

The heteroatoms present in heteroaryl include the oxidized forms such asNO, SO, and SO₂.

As used herein, an “activated ester” refers to an ester group that isreadily displaced by a hydroxyl or an amine group. Exemplary activatedesters include, but are not limited to N-hydroxysuccinimide ester,nitrophenyl (e.g., 2 or 4-nitrophenyl) ester, dinitrophenyl (e.g.,2,4-dinitrophenyl) ester, sulfo-tetraflurophenyl (e.g.,4-sulfo-2,3,5,6-tetrafluorophenyl) ester, pentafluorophenyl ester,nitropyridyl (e.g., 4-nitropyridyl) ester, trifluoroacetate, andacetate.

The term “halide” refers to F, Cl, Br or I. In one embodiment, thehalide is Cl. In one embodiment, the halide is Br. In one embodiment,the halide is I. In one embodiment, the halide is F.

The term “compound” is intended to include compounds for which astructure or formula or any derivative thereof has been disclosed in thepresent invention or a structure or formula or any derivative thereofthat has been incorporated by reference. The term also includes,stereoisomers, geometric isomers, or tautomers. The specific recitationof “stereoisomers,” “geometric isomers,” “tautomers,” “salt” in certainaspects of the invention described in this application shall not beinterpreted as an intended omission of these forms in other aspects ofthe invention where the term “compound” is used without recitation ofthese other forms.

The term “precursor” of a given group refers to any group which may leadto that group by any deprotection, a chemical modification, or acoupling reaction.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomer” refers to compounds which have identicalchemical constitution and connectivity, but different orientations oftheir atoms in space that cannot be interconverted by rotation aboutsingle bonds.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as crystallization, electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds,” John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand 1 or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “protecting group” or “protecting moiety” refers to asubstituent that is commonly employed to block or protect a particularfunctionality while reacting other functional groups on the compound, aderivative thereof, or a conjugate thereof.

An “carboxylic acid protecting group” is a substituent attached to ancarbonyl group that blocks or protects the carboxylic acid functionalityin the compound. Such groups are well known in the art (see for example,P. Wuts and T. Greene, 2007, Protective Groups in Organic Synthesis,Chapter 5, J. Wiley & Sons, NJ). Suitable carboxylic acid protectinggroup include, but are not limited to, alkyl ester (e.g., methyl esteror tert-butyl ester), benzyl ester, thioester (e.g., tert-butylthioester), silyl ester (e.g., trimethylsilyl ester), 9-fluorenylmehtylester, (2-trimethylsilyl)ethoxymethyl ester, 2-(trimethylsilyl)ethylester, diphenylmethyl ester or oxazoline. In certain embodiments, thecarboxylic acid protecting group is methyl ester, tert-butyl ester,benzyl ester or trimethylsilyl ester. In certain embodiments, thecarboxylic acid protecting group is tert-butyl ester.

As used herein, “carboxylic acid deprotecting agent” refers a reagentthat is capable of cleaving a carboxylic acid protecting group to formfree carboxylic acid. Such reagents are well known in the art (see forexample P. Wuts and T. Greene, 2007, Protective Groups in OrganicSynthesis, Chapter 5, J. Wiley & Sons, NJ) and depend on the carboxylicacid protecting group used. For example, when the carboxylic acidprotecting group is tert-butyl ester, it can be cleaved with an acid. Incertain embodiment, the carboxylic acid deprotecting agent istrifluoroacetic acid.

As used herein, “alcohol activating agent” refers a reagent thatincreases the reactivity of a hydroxyl group thereby making the hydroxylgroup a better leaving group. Examples of such alcohol activating agentsinclude p-toluenesulfonyl chloride, thionyl chloride, triflic anhydride,mesyl chloride, mesyl anhydride, triphenylphosphine, acyl chloride,4-dimethylaminopyridine, and others. In certain embodiments, the alcoholactivating agent is thionyl chloride. In certain embodiment, the alcoholactivating agent is triphenylphosphine.

The phrase “salt” as used herein, refers to an organic or inorganicsalts of a compound of the invention. Exemplary salts include, but arenot limited, to sulfate, citrate, acetate, oxalate, chloride, bromide,iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate,lactate, salicylate, acid citrate, tartrate, oleate, tannate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucuronate, saccharate, formate, benzoate,glutamate, methanesulfonate “mesylate,” ethanesulfonate,benzenesulfonate, p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counter ion.The counter ion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a salt may have morethan one charged atom in its structure. Instances where multiple chargedatoms are part of the salt can have multiple counter ions. Hence, a saltcan have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired salt may beprepared by any suitable method available in the art, for example,treatment of the free base with an inorganic acid, such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonicacid, phosphoric acid and the like, or with an organic acid, such asacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid,malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid,a pyranosidyl acid, such as glucuronic acid or galacturonic acid, analpha hydroxy acid, such as citric acid or tartaric acid, an amino acid,such as aspartic acid or glutamic acid, an aromatic acid, such asbenzoic acid or cinnamic acid, a sulfonic acid, such asp-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the compound of the invention is an acid, the desired salt may beprepared by any suitable method, for example, treatment of the free acidwith an inorganic or organic base, such as an amine (primary, secondaryor tertiary), an alkali metal hydroxide or alkaline earth metalhydroxide, or the like. Illustrative examples of suitable salts include,but are not limited to, organic salts derived from amino acids, such asglycine and arginine, ammonia, primary, secondary, and tertiary amines,and cyclic amines, such as piperidine, morpholine and piperazine, andinorganic salts derived from sodium, calcium, potassium, magnesium,manganese, iron, copper, zinc, aluminum and lithium.

In certain embodiments, the salt is a pharmaceutically acceptable salt.The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

Methods of the Present Invention

The present invention provides modular synthetic methods for preparingindolinobenzodiazepine dimer compounds and precursors. The precursorcompounds prepared by the present invention, such as the compound offormula (V) or (V-1) or a salt thereof described below, can be used forsynthesizing indolinobenzodiazepine dimer compounds having diverselinkers for covalent linkage with cell-binding agents to formcell-binding agent-indolinobenzodiazepine conjugates.

In a first embodiment, the present invention provides a method ofpreparing a compound of formula (A):

or a salt thereof, comprising reacting a compound of formula (V):

or a salt thereof, with a compound of formula (X):

wherein:

each double line

between N and C independently represents a single bond or a double bond,provided that when it is a double bond X is absent and Y is —H, and whenit is a single bond, X and Y are both —H; and

E is —OH, halide or —C(═O)E is an activated ester.

Also included in the first embodiment is a method a method of preparinga compound of formula (dA):

or a salt thereof, comprising reacting a compound of formula (dV):

or a salt thereof, with a compound of formula (X):

wherein the variables are the same as described for formula (A).

In certain embodiments, for compounds of formula (A) or (dA), bothdouble line

between N and C independently represent a double bond. In certainembodiments, both double line

between N and C independently represent a single bond.

In certain embodiments, for compounds of formula (A) or (dA), one of thedouble line

between N and C represents a double bond; and the other double line

between N and C represents a single bond, the compound of formula (A) isrepresented by the following formula:

or a salt thereof, and the compound of formula (dA) is represented bythe following formula:

or a salt thereof.

In a second embodiment, for method described in the first embodiment,the compound of formula (A) or a salt thereof is represented by formula(A-1):

or a salt thereof, and the method comprises reacting a compound offormula (V-1):

or a salt thereof, with a compound of formula (X-1):

wherein E is —OH, halide or —C(═O)E is an activated ester.

Also in the second embodiment, for method described in the firstembodiment, the compound of formula (dA) or a salt thereof isrepresented by formula (dA-1):

or a salt thereof, and the method comprises reacting a compound offormula (dV-1):

or a salt thereof, with a compound of formula (X-1):

wherein E is —OH, halide or —C(═O)E is an activated ester.

In a third embodiment, the present invention provides a method ofpreparing a compound of formula (A):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (IV):

or a salt thereof, with a reducing agent to form a compound of formula(V):

or a salt thereof; and

2) reacting the compound of formula (V) or a salt thereof, with acompound of formula (X):

wherein:

each double line

between N and C independently represents a single bond or a double bond,provided that when it is a double bond X is absent and Y is —H, and whenit is a single bond, X and Y are both —H; and

E is —OH, halide or —C(═O)E is an activated ester.

Also included in the third embodiment is a method of preparing acompound of formula (dA):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (dIV):

or a salt thereof, with a reducing agent to form a compound of formula(dV):

or a salt thereof; and

2) reacting the compound of formula (dV) or a salt thereof, with acompound of formula (X):

wherein the variables are the same as described for formula (A).

In certain embodiments, both double line

between N and C independently represent a double bond. In certainembodiments, both double line

between N and C independently represent a single bond.

In certain embodiments, one of the double line

between N and C represents a double bond; and the other double line

between N and C represents a single bond, the compound of formula (A) isrepresented by the following formula:

or a salt thereof, and the compound of formula (dA) is represented bythe following formula:

or a salt thereof.

In a fourth embodiment, for the method of the third embodiment, thecompound of formula (A) or a salt thereof is represented by formula(A-1):

or a salt thereof, and the method comprises the steps of:

1) reacting a compound of formula (IV-1):

or a salt thereof, with a reducing agent to form a compound of formula(V-1):

or a salt thereof; and

2) reacting the compound of formula (V-1) or a salt thereof, with acompound of formula (X-1):

wherein E is —OH, halide or —C(═O)E is an activated ester.

Also in the fourth embodiment, for the method of the third embodiment,the compound of formula (dA) or a salt thereof is represented by formula(dA-1):

or a salt thereof, and the method comprises the steps of:

1) reacting a compound of formula (dIV-1):

or a salt thereof, with a reducing agent to form a compound of formula(dV-1):

or a salt thereof; and

2) reacting the compound of formula (dV-1) or a salt thereof, with acompound of formula (X-1):

wherein E is —OH, halide or —C(═O)E is an activated ester

In a fifth embodiment, for the methods of the third or fourthembodiment, Any suitable reducing reagent that can convert a nitro(—NO₂) group to an amine (—NH₂) group can be used in the reaction ofstep 1). In certain embodiments, the reducing reagent is selected fromthe group consisting of: hydrogen gas, sodium hydrosulfite, sodiumsulfide, stannous chloride, titanium (II) chloride, zinc, iron andsamarium iodide. In certain embodiments, the reducing reagent isFe/NH₄Cl, Fe/NH₄Cl, Zn/NH₄Cl, FeSO₄/NH₄OH, or Sponge Nickel. In specificembodiments, the reducing agent is Fe/NH₄Cl.

In certain embodiments, the reaction between the compound of formula(IV), (dIV), (IV-1) or (dIV-1) with the reducing agent is carried out ina mixture of water and one or more organic solvents. Any suitableorganic solvent can be used. Exemplary organic solvents include, but arenot limited to, DMF, CH₂Cl₂, dichloroethane, THF, dimethylacetamide,methanol, ethanol, etc. In certain embodiments, the organic solvent isTHF or methanol or a combination thereof. In a specific embodiment, thereaction between the compound of formula (IV), (dIV), (IV-1) or (dIV-1)with the reducing agent is carried out in a mixture of water, THF andmethanol.

In a sixth embodiment, for the methods of any one of the first, second,third, fourth or fifth embodiment, E is —OH and the reaction between thecompound of formula (V) and the compound of formula (X), between thecompound of formula (dV) and the compound of formula (X), between thecompound of formula (V-1) and (X-1), or between the compound of formula(dV-1) and (dX-1) is carried out in the presence of an activating agent.

In certain embodiments, the activating agent is a carbodiimide, auronium, an active ester, a phosphonium,2-alkyl-1-alkylcarbonyl-1,2-dihydroquinoline,2-alkoxy-1-alkoxycarbonyl-1,2-dihydroquinoline,2,4,6-trialkyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide, oralkylchloroformate. In a specific embodiment, the activating agent is acarbodiimide. In a more specific embodiment, the activating agent isdicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), ordiisopropylcarbodiimide (DIC). In another specific embodiment, theactivating agent is N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ).

In certain embodiments, the reaction between the compound of formula (V)and the compound of formula (X), between the compound of formula (dV)and the compound of formula (X), between the compound of formula (V-1)and (X-1) or between the compound of formula (dV-1) and (X-1) is carriedout in an organic solvent or a solvent mixture. Any suitable organicsolvent described herein can be used. In certain embodiments, theorganic solvent is dichloromethane or methanol or a mixture thereof.

In a seventh embodiment, the present invention provides a methodpreparing a compound of formula (Xa):

or a salt thereof, comprising reacting the compound of formula (IX):

or a salt thereof, with a carboxylic acid deprotecting agent, wherein P₁is a carboxylic acid protecting group.

In an eighth embodiment, for the method of the seventh embodiment, thecompound of formula (Xa) is represented by formula (X-1a):

or a salt thereof, and the method comprises reacting the compound offormula (IX-1):

or a salt thereof, with a carboxylic acid deprotecting agent, wherein P₁is a carboxylic acid protecting group.

In a ninth embodiment, the present invention provides a method ofpreparing a compound of formula (IX):

comprising reacting a compound of formula (VIII):

or a salt thereof, with a compound of formula (c):

or a salt thereof, wherein E₁ is —OH, halide or —C(═O)E₁ is an activatedester; and P₁ is an carboxylic acid protecting group.

In a tenth embodiment, the compound of formula (IX) or a salt thereof isrepresented by formula (IX-1):

or a salt thereof, and the method comprises reacting a compound offormula (VIII):

or a salt thereof, with a compound of formula (c-1):

or a salt thereof, wherein E₁ is —OH, halide or —C(═O)E₁ is an activatedester; and P₁ is a carboxylic acid protecting group.

In a eleventh embodiment, the present invention provides a method ofpreparing a compound of formula (Xa):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (VIII):

or a salt thereof, with a compound of formula (c):

or a salt thereof, to form a compound of formula (IX):

and

2) reacting the compound of formula (IX) with a carboxylic aciddeprotecting agent, wherein E₁ is —OH, halide or —C(═O)E₁ is anactivated ester; and P₁ is a carboxylic acid protecting group.

In a twelfth embodiment, the compound of formula (Xa) or a salt thereofis represented by formula (X-1a):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (VIII):

or a salt thereof, with a compound of formula (c-1):

or a salt thereof, to form a compound of formula (IX-1):

and

2) reacting the compound of formula (IX-1) with a carboxylic aciddeprotecting agent, wherein E₁ is —OH, halide or —C(═O)E₁ is anactivated ester; and P₁ is a carboxylic acid protecting group.

In certain embodiments, for the method of the seventh, eighth, ninth,tenth, eleventh or twelfth embodiment, the carboxylic acid protectinggroup represented by P₁ can be any suitable carboxylic acid protectinggroup known in the art. In certain embodiments, the carboxylic acidprotecting group include, but are not limited to alkyl ester (e.g.,methyl ester or tert-butyl ester), benzyl ester, thioester (e.g.,tert-butyl thioester), silyl ester (e.g., trimethylsilyl ester),9-fluorenylmehtyl ester, (2-trimethylsilyl)ethoxymethyl ester,2-(trimethylsilyl)ethyl ester, diphenylmethyl ester or oxazoline. Incertain embodiments, the carboxylic acid protecting group is methylester, tert-butyl ester, benzyl ester or trimethylsilyl ester, i.e., P₁is —OMe, —O^(t)Bu, —OBn, —O-silyl (e.g., —OSi(Me)₃). In certainembodiments, the carboxylic acid protecting group is tert-butyl ester,i.e., P₁ is —O^(t)Bu.

To deprotect the carboxylic acid protecting group, any suitabledeprotecting agent known in the art can be used. The suitabledeprotecting agent depends on the identity of the carboxylic acidprotecting group. For example, when P₁ is —O^(t)Bu, the protecting groupcan be removed by the treatment with an acid, a base or a suitablereductant. In certain embodiments, an acid can be used to remove thetert-butyl ester protecting group. Exemplary acids include, but are notlimited to, formic acid, acetic acid, trifluoroacetic acid, hydrochloricacid, and phosphoric acid. In a specific embodiment, trifluoroaceticacid is used as the deprotecting agent.

In certain embodiments, the deprotection reaction can be carried in anysuitable organic solvent(s). Exemplary organic solvents include, but arenot limited to, DMF, CH₂Cl₂, dichloroethane, THF, dimethylacetamide,methanol, ethanol, etc. In certain embodiments, the deprotectionreaction is carried out in dichloromethane.

In certain embodiments, for method of the ninth, tenth, eleventh ortwelfth embodiment, E₁ is —OH and the reaction between the compound offormula (VIII) and the compound of formula (c) or the compound offormula (c-1) is carried out in the presence of an activating agent.

In certain embodiments, the activating agent is a carbodiimide, auronium, an active ester, a phosphonium,2-alkyl-1-alkylcarbonyl-1,2-dihydroquinoline,2-alkoxy-1-alkoxycarbonyl-1,2-dihydroquinoline,2,4,6-trialkyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide, oralkylchloroformate. In a specific embodiment, the activating agent is2,4,6-trialkyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide. In amore specific embodiment, the activating agent is2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide. Incertain embodiments, the reaction between the compound of formula (VIII)and the compound of formula (c) or the compound of formula (c-1) iscarried out in the presence of a base. In certain embodiments, the baseis a non-nucleophilic base. Exemplary non-nucleophilic bases include,but are not limited to, triethylamine, imidazole, diisopropylethylamine,pyridine, 2,6-lutidine, dimethylformamide,1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), or tetramethylpiperidine. In aspecific embodiment, the base is triethylamine or diisopropylethylamine.In another specific embodiment, the base is diisopropylethylamine.

In certain embodiments, the reaction between the compound of formula(VIII) and the compound of formula (c) or the compound of formula (c-1)is carried out in the presence of an activating agent described aboveand a base described above. In certain embodiments, the reaction iscarried out in the presence of2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide andtriethylamine or diisopropylethylamine. In a specific embodiment, thereaction is carried out in the presence of2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide anddiisopropylethylamine.

Any suitable organic solvents can be used for the reaction the reactionbetween the compound of formula (VIII) and the compound of formula (c)or the compound of formula (c-1). In certain embodiments, the reactionis carried out in dichloromethane.

In a thirteenth embodiment, for the method of the ninth, tenth,eleventh, or twelfth embodiment, the compound of formula (VIII) isrepresented by formula (VIIIa):

and the compound of formula (VIIIa) or a salt thereof is prepared by amethod comprising the steps of:

a) reacting a compound of formula (VI):

or a salt thereof, with a compound of formula (d):

or a salt thereof, to form a compound of formula (VII):

and

b) reacting the compound of formula (VII) with a carboxylic aciddeprotecting agent to form the compound of formula (VIIIa) or a saltthereof, wherein P₂ is a carboxylic acid protecting group.

Any suitable carboxylic acid protecting group can be used. In certainembodiments, the carboxylic acid protecting group include, but are notlimited to alkyl ester (e.g., methyl ester or tert-butyl ester), benzylester, thioester (e.g., tert-butyl thioester), silyl ester (e.g.,trimethylsilyl ester), 9-fluorenylmehtyl ester,(2-trimethylsilyl)ethoxymethyl ester, 2-(trimethylsilyl)ethyl ester,diphenylmethyl ester or oxazoline. In certain embodiments, thecarboxylic acid protecting group is methyl ester, tert-butyl ester,benzyl ester or trimethylsilyl ester, i.e., P₂ is —OMe, —O^(t)Bu, —OBn,—O-silyl (e.g., —OSi(Me)₃). In certain embodiments, the carboxylic acidprotecting group is tert-butyl ester, i.e., P₂ is —O^(t)Bu.

To deprotect the carboxylic acid protecting group, any suitablecarboxylic deprotecting agent known in the art can be used. Suitabledeprotecting agents depend on the identity of the carboxylic acidprotecting group. For example, when P₂ is —O^(t)Bu, the protecting groupcan be removed by the treatment with an acid, a base or a suitablereductant. In certain embodiments, an acid can be used to remove thetert-butyl ester protecting group. Exemplary acids include, but are notlimited to, formic acid, acetic acid, trifluoroacetic acid, hydrochloricacid, and phosphoric acid. In a specific embodiment, trifluoroaceticacid is used as the carboxylic acid deprotecting agent.

In certain embodiments, the deprotection reaction can be carried in anysuitable organic solvent(s). Exemplary organic solvents include, but arenot limited to, DMF, CH₂Cl₂, dichloroethane, THF, dimethylacetamide,methanol, ethanol, etc. In certain embodiments, the deprotectionreaction is carried out in dichloromethane.

In certain embodiments, the reaction between the compound of formula(VI) and the compound of formula (d) is carried out in the presence ofan activating agent. Any suitable activating agent described herein canbe used. In certain embodiments, the activating agent is2,4,6-trialkyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide. In amore specific embodiment, the activating agent is2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide.

Any suitable organic solvents can be used for the reaction the reactionbetween the compound of formula (VI) and the compound of formula (d). Incertain embodiments, the reaction is carried out in dichloromethane.

In a fourteenth embodiment, the present invention provides a method ofpreparing a compound of formula (II),

comprising reacting a compound of formula (I):

with hydrochloric acid in toluene.

Also included in the fourteenth embodiment is a method of preparing acompound of formula (dII),

comprising reacting a compound of formula (dI):

with hydrochloric acid in toluene.

In a fifteenth embodiment, the present invention provides a method ofpreparing a compound of formula (IV-1):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (I):

with hydrochloric acid in toluene to form a compound of formula (II):

2) reacting the compound of formula (II) with a monomer compound offormula (a),

to form a compound of formula (III):

or a salt thereof;

3) reacting the compound of formula (III) or a salt thereof with amonomer compound of formula (b):

to form the compound of formula (IV-1) or a salt thereof.

Also included in the fifteenth embodiment is a method of preparing acompound of formula (dIV-1):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (dI):

with hydrochloric acid in toluene to form a compound of formula (dII):

2) reacting the compound of formula (dII) with a monomer compound offormula (a),

to form a compound of formula (dIII):

or a salt thereof;

3) reacting the compound of formula (dIII) or a salt thereof with amonomer compound of formula (b):

to form the compound of formula (dIV-1) or a salt thereof.

In a sixteenth embodiment, the present invention provides a method ofpreparing a compound of formula (A-1):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (I):

with hydrochloric acid in toluene to form a compound of formula (II):

2) reacting the compound of formula (II) with a monomer compound offormula (a),

to form a compound of formula (III):

or a salt thereof;

3) reacting the compound of formula (III) or a salt thereof with amonomer compound of formula (b):

to form a compound of formula (IV-1):

or a salt thereof;

4) reacting the compound of formula (IV-1) or a salt thereof with areducing agent to form a compound of formula (V-1):

or a salt thereof; and

5) reacting the compound of formula (V-1) or a salt thereof, with acompound of formula (X-1):

to form the compound of formula (A-1) or a salt thereof, wherein E is—OH, halide or —C(═O)E is an activated ester.

Also included in the sixteenth embodiment is a method of preparing acompound of formula (dA-1):

or a salt thereof, comprising the steps of:

1) reacting a compound of formula (dI):

with hydrochloric acid in toluene to form a compound of formula (dII):

2) reacting the compound of formula (dII) with a monomer compound offormula (a),

to form a compound of formula (dIII):

or a salt thereof;

3) reacting the compound of formula (dIII) or a salt thereof with amonomer compound of formula (b):

to form a compound of formula (dIV-1):

or a salt thereof;

4) reacting the compound of formula (dIV-1) or a salt thereof with areducing agent to form a compound of formula (dV-1):

or a salt thereof; and

5) reacting the compound of formula (dV-1) or a salt thereof, with acompound of formula (X-1):

to form the compound of formula (dA-1) or a salt thereof, wherein E is—OH, halide or —C(═O)E is an activated ester.

In a seventeenth embodiment, for the method of the fourteenth, fifteenthor sixteenth embodiment, the compound of formula (I) or (dI) is reactedwith concentrated hydrochloric acid to form the compound of formula (II)or (dII) respectively. For example, 30-38 w/w % of hydrochloric acid canbe used.

In certain embodiments, the reaction between the compound of formula (I)or (dI) and hydrochloric acid is carried out at a temperature between30° C. and 110° C., between 40° C. and 105° C., between 50° C. and 100°C., between 60° C. and 100° C., between 70° C. and 100° C., between 80°C. and 100° C. or between 90° C. and 100° C. In certain embodiments, thereaction is carried out at 95° C.

The reaction between the compound of formula (I) or (dI) andhydrochloric acid can be carried out until the reaction is insubstantial completion. For example, the reaction can be carried outbetween 5 minutes to 1 week, between 5 minutes to 72 hours, between 1hour to 48 hours, between 1 hour to 12 hours, between 6 hours to 18hours, or between 1 hour to 6 hours.

In certain embodiments, the compound of formula (II) or (dII) obtainedfrom the reaction of the compound of formula (I) or (dI) andhydrochloric acid is purified. The compound of formula (II) or (dII) canbe purified by column chromatography or crystallization. In certainembodiments, the compound of formula (II) or (dII) is purified bycrystallization. In a specific embodiment, the compounds of formula (II)or (dII) is crystallized in toluene. For example, the compound offormula (II) or (dII) is crystallized by dissolving the compound in hottoluene followed by cooling until the compound crystallized out thesolution.

In a eighteenth embodiment, for methods of the fifteenth, sixteenth orseventeenth embodiment, the compound of formula (II) or (dII) is reactedwith the monomer compound of formula (a) in the presence of an alcoholactivating agent. Any suitable alcohol activating agent can be used. Incertain embodiments, the alcohol activating agent is atrialkylphosphine, triarylphosphine, or triheteroarylphosphine. In aspecific embodiment, the alcohol activating agent is trimethylphosphine,tributylphosphine, tri(o-tolyl)phosphine, tri(m-tolyl)phosphine,tri(p-tolyl)phosphine, tri(2-pyridyl)phosphine, tri(3-pyridyl)phosphine,tri(4-pyridyl)phosphine, or[4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl)phenyl]diphenylphosphine. In another embodiment, the alcohol activating agentcan be a phosphine-like reagent, such as(tributylphosphoranylidene)acetonitrile,(cyanomethylene)tributylphosphorane (CMBP), or(cyanomethylene)trimethylphosphorane (CMMP). In a more specificembodiment, the alcohol activating agent is triphenylphosphine. In yetanother more specific embodiment, the alcohol is tributylphosphine. Inone embodiment, the alcohol activating agent can be polymer-bound orpolymer-supported, such as polymer-bound or polymer-supported trialkylphosphine, triarylphosphine (e.g., triphenylphosphine), ortriheteroarylphosphine.

In certain embodiments, for the method described in the eighteenthembodiment, the compound of formula (II) or (dII) is reacted with themonomer compound of formula (a) the presence of an azodicarboxylate. Inone embodiment, the azodicarboxylate is selected from the groupconsisting of: diethyl azodicarboxylate (DEAD), diisopropylazodicarboxylate (DIAD), 1,1′-(azodicarbonyl)dipiperidine (ADDP),ditertbutyl azodicarboxylate (DTAD),1,6-dimethyl-1,5,7-hexahydro-1,4,6,7-tetrazocin-2,5-dione (DHTD),di-(4-chlorobenzyl)azodicarboxylate (DCAD), azodicarboxylicdimorpholide, N,N,N′,N′-tetramethylazodicarboxamide (TMAD),N,N,N′,N′-tetraisopropylazodicarboxamide (TIPA), 4,4′-azopyridine, bis(2,2,2-trichloroethyl) azodicarboxylate,o-(tert-Butyldimethylsilyl)-N-tosylhydroxylamine,di-(4-chlorobenzyl)azodicarboxylate, cyclic1,6-dimethyl-1,5,7-hexahydro-1,4,6,7-tetrazocin-2,5-dione (DHTD),dimethyl acetylenedicarboxylate (DMAD), di-2-methoxyethylazodicarboxylate, di-(4-chlorobenzyl)azodicarboxylate andbis(4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl) azodicarboxylate. Morespecifically, the azodicarboxylate is DIAD. In one embodiment, theazodicarboxylate is polymer-bound or polymer supported, such aspolymer-supported alkylazodicarboxylate (e.g. polymer-bound DEAD, DIAD,DTAD or ADDP).

In a specific embodiment, for methods of the eighteenth embodiment, thecompound of formula (II) or (dII) is reacted with the monomer compoundof formula (a) in the presence of tributylphosphine ortriphenylphosphine and an azodicarboxylate. In one embodiment, theazodicarboxylate is selected from the group consisting of: diethylazodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD),1,1′-(azodicarbonyl)dipiperidine (ADDP), and ditertbutylazodicarboxylate (DTAD). More specifically, the azodicarboxylate isDIAD. In a more specific embodiment, the compound of formula (II) or(dII) is reacted with the monomer compound of formula (a) in thepresence of tributylphosphine and DIAD.

In certain embodiments, the alcohol activating agent and theazodicarboxylate is mixed together to form an alcohol activatingagent-azodicarboxylate complex. The compound of formula (II) or (dII) ismixed with the complex first before contacting with the monomer compoundof formula (a).

In certain embodiments, the reaction of the eighteenth embodimentdescribed above can be carried out in an organic solvent(s). Anysuitable organic solvent(s) described herein can be used. In certainembodiments, the organic solvent is THF.

In a nineteenth embodiment, for the method of the fifteenth, sixteenth,seventeenth, or eighteenth embodiment, in step 3) of the method, thecompound of formula (III) or (dIII) or a salt thereof is reacted withthe monomer compound of formula (b) in the presence of a base. Incertain embodiments, the base is sodium carbonate, potassium carbonate,cesium carbonate, sodium hydride, or potassium hydride. Preferably, thebase is potassium carbonate.

In certain embodiments, the reaction between the compound of formula(III) or (dIII) or a salt thereof and the monomer compound of formula(b) further comprises potassium iodide.

In certain embodiments, the reaction between the compound of formula(III) or (dIII) or a salt thereof and the monomer compound of formula(b) is carried out in the presence of potassium carbonate and potassiumiodide.

Any suitable organic solvents can be used for the methods of thetwentieth embodiment. In one embodiment, the solvent is a polar aproticsolvent. Exemplary solvents include, but are not limited to,dimethylformamide (DMF), CH₂Cl₂, dichloroethane, THF, dimethylacetamide,etc. In certain embodiments, dimethylformamide or dimethylacetamide isused as the solvent.

In a twentieth embodiment, for the method of the fifteenth, sixteenth,seventeenth, eighteenth, or nineteenth embodiment, in the reaction ofstep 4), the reducing reagent is selected from the group consisting of:hydrogen gas, sodium hydrosulfite, sodium sulfide, stanneous chloride,titanium (II) chloride, zinc, iron and samarium iodide. In certainembodiments, the reducing reagent is Fe/NH₄Cl, Fe/NH₄Cl, Zn/NH₄Cl,FeSO₄/NH₄OH, or Sponge Nickel. In specific embodiments, the reducingagent is Fe/NH₄Cl.

In certain embodiments, the reaction between the compound of formula(IV-1) or (dIV-1) with the reducing agent is carried out in a mixture ofwater and one or more organic solvents. Any suitable organic solvent canbe used. Exemplary organic solvents include, but are not limited to,DMF, CH₂Cl₂, dichloroethane, THF, dimethylacetamide, methanol, ethanol,etc. In certain embodiments, the organic solvent is THF or methanol or acombination thereof. In a specific embodiment, the reaction between thecompound of formula (IV-1) or (dIV-1) with the reducing agent is carriedout in a mixture of water, THF and methanol.

In some embodiments, for the methods described above, the compound offormula (dIII) can be prepared by an alternative process comprising thesteps of:

a) introducing an alcohol protecting group onto one of the primaryalcohols of a compound of formula (dI):

by reacting the compound of formula (dI) with an alcohol protectingagent to form a compound of formula (dI1):

b) reacting the compound of formula (dI1) with a chlorinating agent toform a compound of formula (dI2):

c) reacting the compound of formula (dI2) with an alcohol deprotectingagent to form a compound of formula (dI3):

and

d) reacting the compound of formula (dI3) with a sulfonating agent toform a compound of formula (dI4):

and

e) reacting the compound of (dI4) with a monomer compound of formula(a),

to form the compound of formula (dIII), wherein P₁ is an alcoholprotecting group and X₁ is a sulfonate ester.

In one embodiment, P₁ is a silyl protecting group. Exemplary silylprotecting group include, but are not limited to dimethylisopropylsilyl,diethylisopropylsilyl, dimethylhexylsilyl, trimethylsilyl,triisopropylsilyl, tribenzylsilyl, triphenylsilyl,2-norbornyldimethylsilyl, tert-butyldimethylsilyl,tert-butyldiphenylsilyl, 2-trimethyethylsilyl (TEOC), or[2-(trimethylsilyl)ethoxy]methyl. In one embodiment, P₁ is the silylprotecting group is triethylsilyl, triisopropylsilyl, ortert-butyldimethylsilyl. In another embodiment, P₁ istert-butyldimethylsilyl.

In one embodiment, the silyl protecting group is introduced by reactingthe compound of formula (dI) with R—Cl, R—Br, R—I or R—OSO₂CF₃ in thepresence of a base, wherein R is dimethylisopropylsilyl,diethylisopropylsilyl, dimethylhexylsilyl, trimethylsilyl,triisopropylsilyl, tribenzylsilyl, triphenylsilyl,2-norbornyldimethylsilyl, tert-butyldimethylsilyl, ortert-butyldiphenylsilyl. In one embodiment, the base is anon-nucleophilic base, such as imidazole, triethylamine,diisopropylethylamine, pyridine, 2,6-lutidine,1,8-diazabicycloundec-7-ene, or tetramethylpiperidine.

In one embodiment, the chlorinating reagent is selected from the groupconsisting of carbon tetrachloride, methanesulfonyl chloride, sulfurylchloride, thionyl chloride, cyanuric chloride, N-chlorosuccinimide,phosphorus (V) oxychloride, phosphorus pentachloride, and phosphorustrichloride. In one embodiment, the chlorinating reagent ismethanesulfonyl chloride.

In one embodiment, the alcohol deprotecting reagent istetra-n-butylammonium fluoride, tris(dimethylamino)sulfoniumdifluorotrimethylsilicate, hydrogen fluoride or a solvate thereof,hydrogen fluoride pyridine, silicon tetrafluoride, hexafluorosilicicacid, cesium fluoride, hydrochloric acid, acetic acid, trifluoroaceticacid, pyridinium p-toluensulfonate, p-toluenesulfonic acid (p-TsOH),formic acid, periodic acid. In one embodiment, the alcohol deprotectingreagent is hydrogen fluoride pyridine.

In one embodiment, X₁ is mesylate, tosylate, brosylate, or triflate. Inanother embodiment, X₁ is mesylate.

In one embodiment, the sulfonating reagent is methanesulfonyl anhydride,methanesulfonyl chloride, p-toluenesulfonyl chloride,4-bromobenzenesulfonyl chloride, or trifluoromethanesulfonyl anhydride.In one embodiment, the sulfonating reagent is methanesulfonyl anhydride.

In one embodiment, the alternative process for making the compound offormula (dIII) comprises the steps of:

a) reacting the compound of formula (dI) withtert-butylchlorodimethylsilane to form a compound of formula (dI1′):

b) reacting the compound of formula (dI1′) with methanesulfonyl chlorideto form a compound of formula (dI2′):

c) reacting the compound of formula (dI2′) with an alcohol deprotectingagent to form a compound of formula (dI3):

wherein the alcohol deprotecting agent is HF-pyridine;

d) reacting the compound of formula (dI3) with methanesulfonic anhydrideor methanesulfonyl chloride to form a compound of formula (dI4′):

and

-   -   e) reacting the compound of (dI4′) with a monomer compound of        formula (a),

in the presence of a base (e.g., sodium carbonate or potassiumcarbonate) to form the compound of formula (dIII).

In a twenty-first embodiment, for the method of the sixteenth,seventeenth, eighteenth, nineteenth, or twentieth embodiment, thecompound of formula (X-1) is represented by formula (X-1a):

and the compound of formula (X-1a) or a salt thereof is prepared by amethod comprising the steps of:

a) reacting a compound of formula (VI):

or a salt thereof, with a compound of formula (d):

or a salt thereof, to form a compound of formula (VII):

or a salt thereof;

b) reacting the compound of formula (VII) or a salt thereof with acarboxylic acid deprotecting agent to form a compound of formula(VIIIa):

or a salt thereof;

c) reacting the compound of formula (VIIIa) or a salt thereof with acompound of formula (c-1):

or a salt thereof, to form a compound of formula (IX-1):

or a salt thereof; and

d) reacting the compound of formula (IX-1) or a salt thereof with acarboxylic acid deprotecting agent, wherein P₁ and P₂ are eachindependently a carboxylic acid protecting group.

In certain embodiments, for the method of the twenty-second embodiment,P₁ and P₂ are each independently a suitable carboxylic acid protectinggroup described herein. In certain embodiments, P₁ and P₂ are eachindependently —OMe, —O^(t)Bu, —OBn, —O-silyl (e.g., —OSi(Me)₃). Incertain embodiments, P₁ and P₂ are both —O^(t)Bu.

To deprotect the carboxylic acid protecting group, any suitablecarboxylic deprotecting agent known in the art can be used. Suitabledeprotecting agents depend on the identity of the carboxylic acidprotecting group. For example, when P₁ and P₂ are —O^(t)Bu, theprotecting group can be removed by the treatment with an acid, a base ora suitable reductant. In certain embodiments, an acid can be used toremove the tert-butyl ester protecting group. Exemplary acids include,but are not limited to, formic acid, acetic acid, trifluoroacetic acid,hydrochloric acid, and phosphoric acid. In a specific embodiment,trifluoroacetic acid is used as the carboxylic acid deprotecting agent.

In certain embodiments, the deprotection reaction can be carried in anysuitable organic solvent(s). Exemplary organic solvents include, but arenot limited to, DMF, CH₂Cl₂, dichloroethane, THF, dimethylacetamide,methanol, ethanol, etc. In certain embodiments, the deprotectionreaction is carried out in dichloromethane.

In certain embodiments, for the method of the twenty-second embodiment,the reaction between the compound of formula (VI) and the compound offormula (d) and the reaction between the compound of formula (VIIIa) andthe compound of formula (c-1) are carried out in the presence of anactivating agent. In certain embodiments, the activating agent isindependently selected from a2,4,6-trialkyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide,carbodiimide, a uronium, an active ester, a phosphonium,2-alkyl-1-alkylcarbonyl-1,2-dihydroquinoline,2-alkoxy-1-alkoxycarbonyl-1,2-dihydroquinoline, and alkylchloroformate.In certain embodiments, the activating agent is a2,4,6-trialkyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide. In aspecific embodiment, the activating agent is2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide.

Any suitable organic solvents can be used for the reaction between thecompound of formula (VI) and the compound of formula (d) or between thecompound of formula (VIIIa) and the compound of formula (c-1). Incertain embodiments, the reaction is carried out in dichloromethane.

Compounds of the Invention

Also provided in the present invention is compounds described herein,e.g., compounds of formula (A), (dA), (A′), (dA′), (A-1), (dA-1), (II),(dII), (III), (dIII), (IV), (dIV), (IV-1), (dIV-1), (V), (dV), (V-1),(dV-1), (VI), (VI-1), (VII), (VIII), (VIIIa), (IX), (IX-1), (X), (X-1),(Xa), or (X-1a) or a salt thereof.

In certain embodiment, the compound of the present invention isrepresented by formula (VII), (VIII), (VIIIa), (IX-1), (X-1) or (X-1a)or a salt thereof.

In certain embodiments, the compounds described herein, such ascompounds of formula (A), (A′), (A-1), (II), (III), (IV), (IV-1), (V),(V-1), (VI), (VI-1), (VII), (VIII), (VIIIa), (IX), (IX-1), (X), (X-1),(Xa), or (X-1a) or a salt thereof, are isotopically labeled orradio-labeled. Radio-labeled compounds of the compounds described hereincould be useful in radio-imaging, in in vitro assays or in in vivoassays. “Isotopically labeled” or “radio-labeled” compounds areidentical to compounds disclosed herein, but for the fact that one ormore atoms are replaced or substituted by an atom having an atomic massor mass number different from the atomic mass or mass number typicallyfound in nature (i.e., naturally occurring). Any atom in the compoundsof the disclosure not specifically labelled as an isotope is meant torepresent the given element at about its natural isotopic abundance. Forexample, H represents protium (¹H) with a natural abundance of 99.985%and deuterium (²H) with a natural abundance of 0.015%. Suitableradionuclides that may be incorporated in compounds include, but are notlimited to, ²H (also written as D for deuterium), ³H (also written as Tfor tritium) ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl,⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸²Br, ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I. In some embodiments,the radionuclide is ³H, ¹⁴C, ³⁵S, ⁸²Br, or ¹²⁵I. In some embodiments,the radionuclide is ³H or ¹²⁵I. While the natural isotopic abundance mayvary in a synthesized compound based on the reagents used, theconcentration of naturally abundant stable hydrogen isotopes such asdeuterium is negligible compared to the concentration of stable isotopein the compounds of Formulae (dA), (dA′), (dA-1), (dII), (dIII), (dIV),(dIV-1), (dV), and (dV-1). Thus, when a particular position of thecompounds of Formulae (dA), (dA′), (dA-1), (dII), (dIII), (dIV),(dIV-1), (dV), and (dV-1), contains a deuterium atom, the concentrationof deuterium at that position is substantially greater than the naturalabundance of deuterium, which is 0.015%. In some embodiments, a positioncontaining a deuterium atom has a deuterium enrichment or deuteriumincorporation or deuterium concentration of at least 1%, of at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%. The term “deuterium enrichment” refers tothe percentage of incorporation of deuterium at a given position of thecompounds of the disclosure in replacement of protium. Synthetic methodsfor incorporating radio-isotopes into organic compounds are applicableto compounds of the invention and are well known in the art. Examples ofsynthetic methods for the incorporation of tritium into target moleculesare catalytic reduction with tritium gas, reduction with sodiumborohydride or reduction with lithium aluminum hydride or tritium gasexposure labeling. Examples of synthetic methods for the incorporationof ¹²⁵I into target molecules are Sandmeyer and like reactions, or arylor heteroaryl bromide exchange with ¹²⁵I.

All references cited herein and in the examples that follow areexpressly incorporated by reference in their entireties.

EXAMPLES

The invention will now be illustrated by reference to non-limitingexamples. Unless otherwise stated, all percentages, ratios, parts, etc.are by weight. All reagents were purchased from the Aldrich ChemicalCo., New Jersey, or other commercial sources. Nuclear Magnetic Resonance(¹H NMR) spectra were acquired on a Bruker 400 MHz instrument. Massspectra were acquired on a Bruker Daltonics Esquire 3000 instrument andLCMS were acquired on an Agilent 1260 Infinity LC with an Agilent 6120single quadrupole MS using electrospray ionization (column: AgilentPoroshell 120 C18, 3.0×50 mm, 2.7 μm, 8 min method: flow rate 0.75mL/min, solvent A: water with 0.1% formic acid, solvent B: MeCN, 5% to98% of MeCN over 7 min and 98% MeCN for 1 min) and UPLC were acquired ona Waters, Acquity system with a single quadrupole MS Zspray™ (column:Acquity BEH C18, 2.1×50 mm, 1.7 μm, method: 2.5 min, flow rate 0.8mL/min, solvent A: water, solvent B: MeCN, 5 to 95% of MeCN over 2.0 minand 95% MeCN for 0.5 min).

The following solvents, reagents, protecting groups, moieties and otherdesignations may be referred to by their abbreviations in parenthesis:

Me=methyl; Et=ethyl; Pr=propyl; i-Pr=isopropyl; Bu=butyl;t-Bu=tert-butyl; Ph=phenyl, and Ac=acetyl

Ala=alanine

aq=aqueous

Bn=benzyl

DCM or CH₂Cl₂=dichloromethane

DIEA or DIPEA=N,N-diisopropylethylamine

DMA=N,N-dimethylacetamide

EEDQ=N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline

EtOAc=ethylacetate

g=grams

h=hour

LC=liquid chromatography

LCMS=liquid chromatography mass spectrometry

min=minutes

mg=miligrams

mL=mililiters

mmol=milimoles

Me=methyl

MeOH=methanol

MS=mass spectrometry

MTBE=Methyl tert-butyl ether

NMR=nuclear magnetic resonance spectroscopy

RT or rt=room temperature (ambient, about 25° C.)

sat or sat′d=saturated

T3P=2,4,6-trialkyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide

TFA=trifluoroacetic acid

THF=tetrahydrofuran

TLC=thin layer chromatography

UPLC=Ultra Performance Liquid Chromatography

Example 1. Synthesis of (S)-tert-butyl2-((S)-2-(6-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-6-oxohexanamido)propanamido)propanoate(compound 1) Step 1. Synthesis of tert-butyl6-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-6-oxohexanoate

To a solution of 1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride(0.288 g, 1.632 mmol) in dichloromethane (DCM) (5 ml, 17 vol) was addedDIPEA (0.777 ml, 4.45 mmol), followed by 6-(tert-butoxy)-6-oxohexanoicacid (0.300 g, 1.483 mmol) as a solution in DCM (5 mL, 17 vol). Letreaction stir at RT and then charged2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide (T3P)(1.781 mL, 2.225 mmol) into the reaction. The reaction was stirred at RTuntil consumption of starting materials (2 h). The reaction was quenchedwith water (10 mL, 34 vol), layers separated and the aqueous layer wasextracted once with DCM (10 mL, 34 vol). The combined organic layerswere washed with sat′d NaHCO₃ (10 mL, 34 vol), brine (10 mL, 34 vol),dried over MgSO₄ and filtered. The filtrate was concentrated undervacuum and the resulting light brown oil was purified by silica gelchromatography (hexane to 100% EtOAc in 20 min,). Fractions containingproduct were combined and concentrated under vacuum and placed in vacuumto dry for 24 hours to obtain desired product, compound 1 (0.409 g,88.5% yield) desired M/Z=324.38, found M+1=325.4. The proton NMR forcompound 1 is shown in FIG. 1.

Step 2. Synthesis of6-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-6-oxohexanoicacid

Compound 1 (0.400 g, 1.233 mmol), was dissolved in a mixture of DCMTFA(1/1 solution 8.0 mL, 20 vol) at RT and was stirred for 60 minutes. Thereaction mixture was concentrated under vacuum, co-evaporated withtoluene (2×5.0 mL, 2×12.5 vol) to obtain compound 2 as a white solid andwas used without further purification (0.331 g, 100% yield). Proton NMRfor compound 2 is shown in FIG. 2.

Step 3. Synthesis of (S)-tert-butyl2-((S)-2-(6-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-6-oxohexanamido)propanamido)propanoate

Compound 2 (0.331 g, 1.233 mmol) was dissolved in DCM (5.0 mL, 15 vol).The mixture was charged with DIPEA (0.3 mL) followed by T3P (1.233 ml,1.603 mmol). A solution of tert-butyl L-alanyl-L-alaninate hydrochloride(312 mg, 1.233 mmol) in DIPEA (0.3 mL) was then added. The reaction wasstirred at rt for 2 h before quenching with water (5.0 mL, 15 vol). Thelayers were separated and the aqueous layer was extracted once with DCM(5.0 mL, 15 vol). The combined organic layers were washed with sat′dNaHCO₃ (5.0 mL, 15 vol) and brine (5.0 mL, 15 vol), dried over MgSO₄,filtered and concentrated under vacuum to give desired product (compound3) as a white/gel like product.

The crude product recrystallized by with hot DCM (3.5 mL, 10 vol),followed by dropwise addition of MTBE (1.0 mL, 3 vol). A white gel likeproduct was formed and filtered to give desired product, compound 3.desired M/Z=466.24, found M+1=467.6. Proton NMR for compound 3 is shownin FIG. 3.

Step 4. Synthesis of(S)-2-((S)-2-(6-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-6-oxohexanamido)propanamido)propanoicacid

Compound 3, was dissolved in DCM (4 mL). TFA (2 mL) was added and thereaction was stirred at room temperature for 2 hours after which thereaction was concentrated under vacuum to give a clear yellow oil. Theoil was co-evaporated with toluene (3×5.0 mL, 3×15 vol).

The oil was then triturated with hot DCM (3.5 mL, 10 vol) and MBTE (1.0mL, 3 vol) was added to deliver a white/yellow solid as the desiredproduct. Solid was dried under vacuum to yield compound 4 (0.350 g,69.2% yield), desired M/Z=410.18, found M+1=411.5.

Example 2. Synthesis of (3-(chloromethyl)-5-nitrophenyl)methanol,Compound 5

In a 250 mL round bottom flask equipped with magnetic stirring, J-Kemfor temperature control, blanket nitrogen, and condenser was chargedwith (5-nitro-1,3-phenylene)dimethanol (5.0 g, 27.3 mmol). Toluene (90.0mL, 18 vol) was added and the resulting suspension was stirred at roomtemperature. To the reaction was charged concentrated hydrochloric acid(37%, 10.0 mL, 2 vol) and the reaction was stirred at room temperaturefor 10 minutes. The reaction was then heated to 95° C. Upon heating to45° C. the reaction became clear with light orange tinge. The reactionwas heated at 95° C. overnight. After stirring at 95° C. overnight, thereaction was cooled to room temperature. The reaction was observed to bebiphasic with small water layer at bottom (˜5.0 mL). The reaction wastransferred to a 250 mL separatory funnel and washed with water (2×50mL, 2×10 vol) followed by saturated sodium bicarbonate (1×50 mL, 1×10vol). The pH of the final wash was 6.0 determined by pH strip. Theorganic phase was retained and concentrated under vacuum to half thevolume (˜50 mL, 10 vol), resulting in slightly hazy solution. Thesolution was stirred in ice/water bath resulting in precipitation. Thesolution was allowed to crystallize at 2° C. for 3 hours. The whitesolid was filtered off under vacuum and was dried under vacuum at 40° C.to for 24 hours to obtain compound 5(3.67 g, 66.0% yield). Desired M/Z201.02 found M−1+2Na 246.00. UPLC retention time: 1.38 min.

Example 3. Synthesis of(S)-9-((3-(chloromethyl)-5-nitrobenzyl)oxy)-8-methoxy-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-one,Compound 6

In a 250 mL round bottom flask equipped with J-Kem, magnetic stirring,nitrogen overlay, and cooling bath was charged monomer (a) (1.0 g, 3.375mmol) and THF (20 mL, 20 vol) and the resulting solution was stirred at19.5° C. (room temperature (RT)). The solution was slightly cloudy withsome undissolved particulates. Compound 5 (0.82 g, 4.05 mmol) was addedto the solution and the resulting mixture was stirred at RT. To themixture was charged tri-n-butylphosphine (0.843 mL, 3.375 mmol) andcooled to 5° C. in ice water bath. The reaction was still cloudy.Diisopropyl azodicarboxylate, DIAD (0.664 mL, 3.375 mmol) was slowlydropped into reaction in a manner so that the exotherm is controlled.The reaction mixture turned clear orange in color (note if reactionturns dark orange/black the rate of addition is too fast). Upon fulladdition, the reaction turned light orange in color. The cooling wasremoved and the reaction was warmed to RT and was stirred overnight.Additional tri-n-butylphosphine (0.169 mL, 0.675 mmol) was added to thereaction and cooled to 5° C. in ice water bath at which point thereaction solution was still cloudy. DIAD (0.133 mL, 0.675 mmol) wasadded dropwise into the reaction in a manner so that the exotherm iscontrolled. Reaction turned clear orange in color (note if reactionturns dark orange/black the rate of addition is too fast). Upon fulladdition reaction turns light orange in color. Removed cooling andreaction was warmed to RT for 1 hour. The reaction mixture wasconcentrated under vacuum and then re-dissolved in dichloromethane (50mL, 50 vol). The resulting dichloromethane solution was washed withwater (2×25 mL, 2×25 vol). The organic phase was retained and slurriedwith potassium carbonate supported silica gel (1.0 g) to removeunreacted monomer (a). The silica gel was removed by filtration usingBuchner funnel under vacuum and the resulting filtrate was concentratedto 5.0 mL (5 vol). The product was purified by silica gel chromatography(0-55% EtOAc/hexanes) to obtain compound 6 (1.0 g, 61.7% yield), DesiredM/Z 479.12, found M+1 480.4. UPLC retention time: 1.88 min.

Example 4. Synthesis of(S)-8-methoxy-9-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-nitrobenzyl)oxy)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-one,Compound 7

In a 250 mL flask equipped with J-Kem, magnetic stirring, nitrogenoverlay, and heating mantle was charged compound 6 (1.0 g, 2.08 mmol).Dry DMA (20 mL, 20 vol) was added and the resulting mixture was stirredat RT resulting in a light brown solution. Monomer (b) (0.644 g, 2.19mmol) was added and the resulting mixture was stirred at RT. KI (0.173g, 1.04 mmol), was then added followed by K₂CO₃ (0.576 g, 4.17 mmol) andthe resulting reaction mixture was stirred at RT and then heated at 35°C. for 4 hours. The reaction was cooled to room temperature and water(20 mL, 20 vol) was added to quench the reaction and precipitate theproduct. Upon water addition reaction is exothermic (20° C. to 40° C.).The resulting mixture was filtered and the solid was washed with water(50 mL, 50 vol). The solid was retained and dissolved in dichloromethane(40 mL, 40 vol) and transferred to a separatory funnel. The organicphase was washed with brine (2×20 mL, 2×20 vol) followed by water (2×20mL, 2×20 vol). The organic phase was retained and concentrated to 10 mL(10 vol) and then slowly added into MTBE (40 mL, 40 vol) resulting inthe formation of a light orange solid in solution. The solution wascooled in ice/water bath and stirred for 1 hour. The solid was filteredunder vacuum dried under vacuum for 24 hours to yield compound 7 (1.6g,). Desired M/Z 737.25, found M+1 738.6. UPLC retention time: 5.89 min.

Example 5

Compound 7 (2.38 g, 3.22 mmol) was dissolved in anhydrous THF (30 mL, 12vol), anhydrous MeOH (4 mL) and water (2.0 mL). Ammonium chloride (1.82g, 10 eq, 32.3 mmol) and iron powder (1.02 g, 16.1 mmol) were added. Themixture was stirred at 60° C. for 3 h while monitoring reaction forcompletion via UPLC.

The reaction mixture was cooled to rt, filtered through Celite andrinsed with DCM (60 mL, 25 vol). The resulting solution was concentratedto dryness on a rotary evaporator and then dissolved in DCM (50 mL, 20vol) and transferred to a separatory funnel. Brine was added (50 mL, 20vol), layers were separated and the organic layer was washed with water(2×25 mL, 2×10 vol). The organic layer was concentrated to dryness (deeporange syrup that foamed a little). The crude product was dissolved inDCM (10 mL, 4 vol) and was slowly dripped into stirring MTBE (50 mL, 20vol). The resulting white slurry as cooled in ice water bath to 2.5° C.and stirred for 1 hour. After 1 hour the solid was filtered under vacuumand washed with MTBE (2×25 mL, 2×10 vol). The solid was dried undervacuum to obtain compound 8 (1.6 g, 70% yield, 80.66% purity by UPLC).

Example 6. Synthesis ofN1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-N6-((S)-1-(((S)-1-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)-5-((((S)-8-methoxy-6-oxo-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)adipamide,Compound 9

To a 50 mL round bottom flask was charged with compound 4 (0.0319 g,0.078 mmol) followed by DCM (3.0 mL, 100 vol). EEDQ was then charged tothe reaction and the resulting mixture was stirred for 5 min. Methanol(0.20 mL, 10 vol) was charged to the reaction to produce a clearsolution. To the reaction solution was charged with a solution ofcompound 8 (50 mg, 0.071 mmol) in DCM (1.0 mL, 30 vol) and the reactionwas stirred at rt for 6 h.

After completion, the reaction was concentrated to 2.0 mL (63 vol). MTBE(4.0 mL, 125 vol) was added to the reaction and white precipitate wasformed. The resulting suspension was stirred for 10 min at rt. The solidwas filtered off to give a white yellow solid which was purified bysilica gel chromatography (100% DCM to 90/10 DCM/MeOH) to yield compound9 (0.037 g, 47.6% yield). UPLC retention time: 5.04 min.

Example 7. Synthesis of Deuterated Compound 8 Step 1: Reduction withBorane-d₃-THF Complex Solution

To a solution of 5-nitroisophthalic acid (0.8 g, 3.79 mmol) intetrahydrofuran (8 ml) was added borane-d₃-THF complex solution (15.16ml, 15.16 mmol) (1M solution, Aldrich, 97.5% D) dropwise at 0° C. Thereaction slowly warmed to room temperature and was stirred 48 hoursuntil consumption of the starting material was complete. After dropwiseaddition of Methanol (8 ml) the mixture was filtered and evaporated. Thedry filtrate was dissolved in ethyl acetate and washed with saturatedsodium bicarbonate, water, and brine. The organic was dried overmagnesium sulfate, filtered and stripped to give compound dI (0.65 g,y=92%). The material was used crude without further purification. ¹H NMR(400 MHz, DMSO-d₆) δ 5.46 (s, 1H), 7.70 (s, 1H), 8.04 (s, 2H)

Step 2

(5-nitro-1,3-phenylene)bis(methan-d2-ol) (0.176 g, 0.938 mmol) (compounddI) was suspended in toluene (3.13 ml). Concentrated hydrochloric acid(0.353 ml, 3.94 mmol) was added dropwise at ambient temperature. Thereaction was then stirred at reflux (95° C.). After 18 hours the mixturewas cooled to ambient temperature and transferred to separatory funnelwith toluene and washed with water (1×15 mL) and aqueous sodiumbicarbonate (1×15 mL). The organic layer was concentrated to dryness toget (3-(chloromethyl-d2)-5-nitrophenyl)methan-d2-ol (0.16 g, y=77%yield) (compound d5) as an off white solid. ¹H NMR (400 MHz, DMSO-d₆) δ5.54 (s, 1H), 7.85 (s, 1H), 8.15 (s, 1H), 8.20 (s, 1H). LCMS: 1.34 minon 2.5 min method.

Step 3

To a solution of(S)-9-hydroxy-8-methoxy-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-one(140 mg, 0.472 mmol) and (3-(chloromethyl-d2)-5-nitrophenyl)methan-d2-ol(121 mg, 0.591 mmol) (compound d5) in anhydrous tetrahydrofuran (2953μl) (stabilized with BHT) was added tri-n-butylphosphine (174 μl, 0.661mmol) under nitrogen at room temperature. The mixture was cooled to 0°C. in an ice bath. After stirring 10 minutes diisopropyl(E)-diazene-1,2-dicarboxylate (139 μl, 0.661 mmol) was added dropwise.The mixture was stirred from 0° C. to room temp over 1 hour upon whichdeionized water (2 mL) was added and stirred for 30 min. The reactionmixture was concentrated to remove tetrahydrofuran, then diluted withdichloromethane and washed with water (2×15 mL). The organic layer wasdried with anhydrous magnesium sulfate, filtered and concentrated.The crude material was purified by silica gel chromatography (ethylacetate/dichloromethane). Fractions containing desired product werecombined and concentrated to give a yellow oil, which was recrystallizedin ethyl acetate/tert-butylmethylether. The resulting solid was filteredand washed with tert-butylmethylether to obtain(S)-9-((3-(chloromethyl-d2)-5-nitrophenyl)methoxy-d2)-8-methoxy-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-one(57 mg, y=40% yield) (compound d6). ¹H NMR (400 MHz, DMSO-d₆) δ 2.90(dd, J=4.2 Hz, 17 Hz, 1H), 3.28 (dd, J=9.6, 12.8 Hz. 1H), 3.48 (dd,J=10.2, 17 Hz, 1H), 3.57 (dd, J=6, 12.8 Hz, 1H), 3.72 (s, 3H), 4.37 (m,1H), 6.37 (d, J=5.6 Hz, 1H), 6.43 (s, 1H), 7.02 (t, J=7.6 Hz, 1H), 7.19(t, J=7.6 Hz, 1H), 7.25 (d, J=7.2 Hz, 1H), 7.31 (s, 1H), 7.99 (s, 1H),8.21 (d, J=8.4 Hz, 1H), 8.32 (s, 2H)

LCMS: 1.84 min on 2.5 min method MS (m/z), found 484.4 (M+1)⁺

Step 4

Potassium iodide (15.44 mg, 0.093 mmol) and anhydrous potassiumcarbonate (51.4 mg, 0.372 mmol) were added to a mixture of(S)-9-((3-(chloromethyl-d2)-5-nitrophenyl)methoxy-d2)-8-methoxy-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-one(90 mg, 0.186 mmol) (compound d6) and(S)-9-hydroxy-8-methoxy-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-onemonomer (57.5 mg, 0.195 mmol) in anhydrous DMA (1860 μl) under nitrogenat ambient temperature. After continuous stirring for 4.5 hours at 35 Cthe reaction mixture was diluted with water and the resulting solid wasfiltered. The solid was re-dissolved in dichloromethane, washed withwater (1×10 mL), dried with anhydrous mag sulfate, filtered andconcentrated. The crude material was re-dissolved in THF/ACN/DI water(3:2:1) and purified by RP-HPLC (Kromasil C18, Acetonitrile/Deionizedwater) Fractions containing desired product were extracted withdichloromethane. The organic extracts were concentrated in vacuo toobtain(S)-8-methoxy-9-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl-d2)-5-nitrophenyl)methoxy-d2)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-one(57 mg, y=41% yield) (compound d7). LCMS: 1.86 min on 2.5 min method MS(m/z), found 742.4 (M+1)⁺

Step 5

(S)-8-methoxy-9-((3-((((S)-8-methoxy-6-oxo-11,12,12a,13-tetrahydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-9-yl)oxy)methyl-d2)-5-nitrophenyl)methoxy-d2)-12a,13-dihydro-6H-benzo[5,6][1,4]diazepino[1,2-a]indol-6-one(57 mg, 0.077 mmol) (compound d7) was suspended in anhydroustetrahydrofuran (1025 μl), anhydrous methanol (342 μl) and deionizedwater. Ammonium chloride (41.1 mg, 0.768 mmol) and iron (21.46 mg, 0.384mmol) were added and the mixture was stirred for two hours at 65 C undernitrogen. The mixture was cooled to room temperature, diluted with 20%methanol/dichloromethane and filtered. The filtrate was concentratedfiltrate and purified by silica gel chromatography(Methanol/Dichloromethane). Fractions containing desired product werecombined and evaporated to obtain compound d8 (44 mg, y=80% yield) as alight yellow solid.

LCMS: 1.62 min on 2.5 min method MS (m/z), found 712.4 (M+1)⁺

Alternatively, Compound of d7 can be Prepared as Follows:

To a solution of compound dI (0.8 g, 4.27 mmol) in anhydrousdichloromethane (30 ml) at 0° C. was addedN-ethyl-N-isopropylpropan-2-amine (1.509 ml, 8.55 mmol) followed bytert-butylchlorodimethylsilane (0.709 g, 4.70 mmol) as a solution inanhydrous N,N-dimethylformamide (5 ml). The reaction was stirred at 0°C. and monitored by TLC (dichloromethane/methanol: 9/1) to give amixture of the starting material, mono and bis-protected products. Afterone hour the reaction was quenched with saturated ammonium chloride, andthen the aqueous solution was extracted with dichloromethane (2×20 ml).The combined organic layers were washed with water (2×50 ml), brine,dried over magnesium sulfate, filtered and stripped to give a crudeyellow oil. The material was then purified by silica gel chromatographyin dichloromethane/methanol to isolate the desired product, compound dII(0.54 g, y=42%)

Compound dII (0.55 g, 1.825 mmol) was dissolved in anhydrousN,N-dimethylformamide (10 ml) and pyridine (0.515 ml, 6.39 mmol) wasadded. The reaction was cooled to 0° C., then methanesulfonyl chloride(0.282 ml, 3.65 mmol) was added dropwise, and reaction stirred for twohours, until completion of starting material. The mixture was firstquenched with saturated sodium bicarbonate, then ethyl acetate was addedand the layer separated. The aqueous layer was extracted with ethylacetate (3×50 ml). The combined organic layers were washed with water,brine, dried over magnesium sulfate and filtered. The solvent wasremoved and the crude compound dIII (0.6 g, y=103%) was used in the nextstep without purification. UPLC=2.27 min (2.5 min method).

To a solution of compound dIII (0.6 g, 1.876 mmol) in anhydroustetrahydrofuran (11.5 ml) was added N,N-Diisopropylethylamine (1.638 ml,9.38 mmol) followed HF-pyridine (0.797 ml, 5.63 mmol) and the reactionwas stirred at room temperature for two hours until completion ofstarting material. The reaction was quenched with saturated sodiumbicarbonate then ethyl acetate was added and layers separated. Theaqueous layer was extracted with ethyl acetate (3×10 ml) and thecombined organic layers were washed with brine, dried over magnesiumsulfate, filtered, and the solvent was removed in vacuo to give compoundd5 that was carried on without purification (0.4 g, y=104%). UPLC=1.36min (2.5 min method). ¹H NMR (400 MHz, DMSO-d₆) δ 5.54 (s, 1H), 7.85 (s,1H), 8.15 (s, 1H), 8.20 (s, 1H)

Compound d5 (400 mg, 1.945 mmol) was dissolved in anhydrousdichloromethane (12.5 ml) and cooled to 0° C. N,N-diisopropylethylamine(1019 μl, 5.84 mmol) was added followed by a solution of methanesulfonicanhydride (439 mg, 2.52 mmol) in dichloromethane. The reaction wasstirred for approximately one hour until completion of the startingmaterial. The reaction was quenched with cold water, the layers wereseparated and the aqueous layer was extracted with DCM (3×20 ml). Thecombined organic layers were washed with water, saturated sodiumbicarbonate, brine, dried over magnesium sulfate and filtered. Theexcess of solvent was removed in vacuo and the crude material was usedin the next step without further purification. UPLC=1.55 min (2.5 minmethod).

To a solution of compound dIV (560 mg, 1.974 mmol) in anhydrousN,N-Dimethylacetamide (18.5 ml) was added potassium carbonate (818 mg,5.92 mmol) followed by a solution of reduced monomer (614 mg, 2.072mmol) in anhydrous N,N-Dimethylacetamide (15 ml). The reaction wasstirred at room temperature for seven hours. Upon completion, thereaction was quenched with water and mixture stirred for ten minutes.The solid was filtered and then dissolved in dichloromethane/methanol(9/1) and washed with brine. The organic layer was separated and driedover magnesium sulfate, filtered and stripped. The crude material waspurified by silica gel chromatography, using hexanes/ethyl acetate togive compound d6 (177 mg, y=18%). MS (m/z): 484.4 (M+1)⁺. UPLC=1.86 min(2.5 min method).

The invention claimed is:
 1. A method of preparing a compound of formula(II),

comprising reacting a compound of formula (I):

with hydrochloric acid in toluene, wherein the hydrochloric acid is30-38 w/w % of hydrochloric acid in water, and wherein the compound offormula (II) is purified by crystallization by cooling a concentratedsolution of the compound in toluene.
 2. A method of preparing a compoundof formula (IV-1):

or a salt thereof, comprising the steps of: 1) reacting a compound offormula (I):

with hydrochloric acid in toluene to form a compound of formula (II),wherein the hydrochloric acid is 30-38 w/w % of hydrochloric acid inwater:

and wherein the compound of formula (II) is purified by crystallizationby cooling a concentrated solution of the compound in toluene; 2)reacting the compound of formula (II) with a monomer compound of formula(a) in the presence of an alcohol activating agent and anazodicarboxylate,

to form a compound of formula (III):

or a salt thereof, wherein the alcohol activating agent is atrialkylphosphine, triarylphosphine, or triheteroarylphosphine, andwherein the azodicarboxylate is selected from the group consisting of:diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD),1,1′-(azodicarbonyl)dipiperidine (ADDP), and ditertbutylazodicarboxylate (DTAD); 3) reacting the compound of formula (III) or asalt thereof with a monomer compound of formula (b) in the presence of abase:

to form the compound of formula (IV-1) or a salt thereof, wherein thebase is sodium carbonate, potassium carbonate, cesium carbonate, sodiumhydride, or potassium hydride.
 3. A method of preparing a compound offormula (A-1):

or a salt thereof, comprising the steps of: 1) reacting a compound offormula (I):

with hydrochloric acid in toluene to form a compound of formula (II),wherein the hydrochloric acid is 30-38 w/w % of hydrochloric acid inwater:

and wherein the compound of formula (II) is purified by crystallizationby cooling a concentrated solution of the compound in toluene; 2)reacting the compound of formula (II) with a monomer compound of formula(a) in the presence of an alcohol activating agent and anazodicarboxylate,

to form a compound of formula (III):

or a salt thereof, wherein the alcohol activating agent is atrialkylphosphine, triarylphosphine, or triheteroarylphosphine, andwherein the azodicarboxylate is selected from the group consisting of:diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD),1,1′-(azodicarbonyl)dipiperidine (ADDP), and ditertbutylazodicarboxylate (DTAD); 3) reacting the compound of formula (III) or asalt thereof with a monomer compound of formula (b) in the presence of abase:

to form a compound of formula (IV-1):

or a salt thereof, wherein the base is sodium carbonate, potassiumcarbonate, cesium carbonate, sodium hydride, or potassium hydride; 4)reacting the compound of formula (IV-1) or a salt thereof with areducing agent to form a compound of formula (V-1):

or a salt thereof; and 5) reacting the compound of formula (V-1) or asalt thereof, with a compound of formula (X-1):

to form the compound of formula (A-1) or a salt thereof, wherein E isOH, halide or —C(═O)E is an activated ester.
 4. The method of claim 1,wherein the reaction between the compound of formula (I) andhydrochloric acid is carried out at a temperature between 40° C. and105° C.
 5. The method of claim 4, wherein the reaction is carried out ata temperature between 90° C. and 100° C.
 6. The method of claim 2,wherein the alcohol activating agent is triphenylphosphine and theazodicarboxylate is diisopropyl azodicarboxylate (DIAD).
 7. The methodof claim 2, wherein the reaction of step 2) comprises the steps of i)mixing the alcohol activating agent and the azodicarboxylate to form analcohol activating agent-azodicarboxylate complex; ii) reacting thecompound of formula (II) with the alcohol activatingagent-azodicarboxylate complex to form a mixture of the compound offormula (II) and the alcohol activating agent-azodicarboxylate complex;and iii) reacting the mixture of step ii) with the monomer compound offormula (a).
 8. The method of claim 2, wherein in step 3), the base ispotassium carbonate.
 9. The method of claim 8, wherein in step 3), thereaction between the compound of formula (III) or a salt thereof and themonomer compound of formula (b) is carried out in the presence ofpotassium iodide.
 10. The method of claim 3, wherein in step 4), thereducing agent is Fe/NH₄Cl.
 11. The method of claim 3, wherein thecompound of formula (X-1) is represented by formula (X-1a):

and the compound of formula (X-1a) or a salt thereof is prepared by amethod comprising the steps of: a) reacting a compound of formula (VI):

or a salt thereof, with a compound of formula (d) in the presence of anactivating agent:

or a salt thereof, to form a compound of formula (VII):

or a salt thereof; b) reacting the compound of formula (VII) or a saltthereof with a carboxylic acid deprotecting agent to form a compound offormula (VIIIa):

or a salt thereof; c) reacting the compound of formula (VIIIa) or a saltthereof with a compound of formula (c-1) in the presence of anactivating agent:

or a salt thereof, to form a compound of formula (IX-1):

or a salt thereof; and d) reacting the compound of formula (IX-1) or asalt thereof with a carboxylic acid deprotecting agent, wherein thecarboxylic acid deprotecting agent is trifluoroacetic acid (TFA),wherein P₁ and P₂ are each independently a carboxylic acid protectinggroup selected from —O^(t)Bu, —OMe, —OBn or —O-silyl, and wherein theactivating agent is a 2,4,6-trialkyl-1,3,5,2,4,6-trioxatriphosphorinane2,4,6-trioxide, carbodiimide, a uronium, an active ester, a phosphonium,2-alkyl-1-alkylcarbonyl-1,2-dihydroquinoline,2-alkoxy-1-alkoxycarbonyl-1,2-dihydroquinoline, or alkylchloroformate.12. The method of claim 11, wherein P₁ and P₂ are both —^(t)Bu.
 13. Themethod of claim 11, wherein the activating agent2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide. 14.The method of claim 2, wherein the reaction between the compound offormula (I) and hydrochloric acid is carried out at a temperaturebetween 40° C. and 105° C.
 15. The method of claim 14, wherein thereaction is carried out at a temperature between 90° C. and 100° C. 16.The method of claim 3, wherein the reaction between the compound offormula (I) and hydrochloric acid is carried out at a temperaturebetween 40° C. and 105° C.
 17. The method of claim 16, wherein thereaction is carried out at a temperature between 90° C. and 100° C. 18.The method of claim 3, wherein the alcohol activating agent istriphenylphosphine and the azodicarboxylate is diisopropylazodicarboxylate (DIAD).
 19. The method of claim 3, wherein in step 3),the base is potassium carbonate.
 20. The method of claim 19, wherein instep 3), the reaction between the compound of formula (III) or a saltthereof and the monomer compound of formula (b) is carried out in thepresence of potassium iodide.